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Pathogenesis associated with a restrictive cardiomyopathy mutant in cardiac troponin T is due to reduced protein stability and greatly increased myofilament Ca2+ sensitivity.
Title
Pathogenesis associated with a restrictive cardiomyopathy mutant in cardiac troponin T is due to reduced protein stability and greatly increased myofilament Ca2+ sensitivity.
Creator
Parvatiyar, Michelle S, Pinto, Jose Renato
Abstract/Description
Dilated and hypertrophic cardiomyopathy mutations in troponin can blunt effects of protein kinase A (PKA) phosphorylation of cardiac troponin I (cTnI), decreasing myofilament Ca2+-sensitivity; however this effect has never been tested for restrictive cardiomyopathy (RCM) mutants. This study explores whether an RCM cardiac troponin T mutant (cTnT-ΔE96) interferes with convergent PKA regulation and if TnT instability contributes to greatly enhanced Ca2+-sensitivity in skinned fibers. Force of...
Show moreDilated and hypertrophic cardiomyopathy mutations in troponin can blunt effects of protein kinase A (PKA) phosphorylation of cardiac troponin I (cTnI), decreasing myofilament Ca2+-sensitivity; however this effect has never been tested for restrictive cardiomyopathy (RCM) mutants. This study explores whether an RCM cardiac troponin T mutant (cTnT-ΔE96) interferes with convergent PKA regulation and if TnT instability contributes to greatly enhanced Ca2+-sensitivity in skinned fibers. Force of contraction in skinned cardiac porcine fiber and spectroscopic studies were performed. A decrease of -0.26 and -0.25 pCa units in Ca2+-sensitivity of contraction after PKA incubation was observed for skinned fibers incorporated with WT or cTnT-ΔE96, respectively. To further assess whether cTnT-ΔE96 interferes solely with transmission of cTnI phosphorylation effects, skinned fibers were reconstituted with PKA pseudo-phosphorylated cTnI (cTnI-SS/DD.cTnC). Fibers displaced with cTnT-WT, reconstituted with cTnI-SS/DD.cTnC decreased Ca2+-sensitivity of force (pCa50=5.61) compared to control cTnI-WT.cTnC (pCa50=5.75), similarly affecting cTnT-ΔE96 (pCa50=6.03) compared to control \cTnI-WT.cTnC (pCa50=6.14). Fluorescence studies measuring cTnC(IAANS) Ca2+-affinity changes due to cTnT-ΔE96 indicated that higher complexity (thin filament) better recapitulates skinned fiber Ca2+ sensitive changes. Circular dichroism revealed reduced α-helicity and earlier thermal unfolding for cTnT-ΔE96 compared to WT. Although ineffective in decreasing myofilament Ca2+-sensitivity to normal levels, cTnT-ΔE96 does not interfere with PKA cTnI phosphorylation mediated effects; 2) cTnT-ΔE96 requires actin to increase cTnC Ca2+-affinity; and 3) deletion of E96 reduces cTnT stability, likely disrupting crucial thin filament interactions. The pathological effect of cTnT-ΔE96 is largely manifested by dramatic myofilament Ca2+-sensitization which still persists even after PKA phosphorylation mediated Ca2+-desensitization.
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Date Issued
2015-02-01
Identifier
FSU_pmch_25450489, 10.1016/j.bbagen.2014.09.029, PMC4276470, 25450489, 25450489, S0304-4165(14)00366-3
Format
Citation
interactive network of long non-coding RNAs facilitates the Drosophila sex determination decision.
Title
An interactive network of long non-coding RNAs facilitates the Drosophila sex determination decision.
Creator
Mulvey, Brett B, Olcese, Ursula, Cabrera, Janel R, Horabin, Jamila I
Abstract/Description
Genome analysis in several eukaryotes shows a surprising number of transcripts which do not encode conventional messenger RNAs. Once considered noise, these non-coding RNAs (ncRNAs) appear capable of controlling gene expression by various means. We find that Drosophila sex determination, specifically the master-switch gene Sex-lethal (Sxl), is regulated by long ncRNAs (>200nt). The lncRNAs influence the dose sensitive establishment promoter of Sxl, SxlPe, which must be activated to specify...
Show moreGenome analysis in several eukaryotes shows a surprising number of transcripts which do not encode conventional messenger RNAs. Once considered noise, these non-coding RNAs (ncRNAs) appear capable of controlling gene expression by various means. We find that Drosophila sex determination, specifically the master-switch gene Sex-lethal (Sxl), is regulated by long ncRNAs (>200nt). The lncRNAs influence the dose sensitive establishment promoter of Sxl, SxlPe, which must be activated to specify female sex. They are primarily from two regions, R1 and R2, upstream of SxlPe and show a dynamic developmental profile. Of the four lncRNA strands only one, R2 antisense, has its peak coincident with SxlPe transcription, suggesting that it may promote activation. Indeed, its expression is regulated by the X chromosome counting genes, whose dose determines whether SxlPe is transcribed. Transgenic lines which ectopically express each of the lncRNAs show they can act in trans, not only impacting the process of sex determination but also altering the levels of the other lncRNAs. Generally, expression of R1 is negative whereas R2 is positive to females. This ectopic expression also results in a change in the local chromatin marks, affecting the timing and strength of SxlPe transcription. The chromatin marks are those deposited by the Polycomb and trithorax groups of chromatin modifying proteins, which we find bind to the lncRNAs. We suggest that the increasing numbers of non-coding transcripts being identified are a harbinger of interacting networks similar to the one we describe.
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Date Issued
2014-09-01
Identifier
FSU_pmch_24954180, 10.1016/j.bbagrm.2014.06.007, PMC4134978, 24954180, 24954180, S1874-9399(14)00159-X
Format
Citation